TAILIEUCHUNG - Báo cáo khoa học: Mixed-type noncompetitive inhibition of anthrax lethal factor protease by aminoglycosides

We report a detailed kinetic investigation of the aminoglycosides neomycin B and neamine as inhibitors of the lethal factor protease fromBacillus anthracis. Both inhibitors display a mixed-type, noncompetitive kinetic pat-tern, which suggests the existence of multiple enzyme–inhibitor binding sites or the involvement of multiple structural binding modes at the same site. | iFEBS Journal Mixed-type noncompetitive inhibition of anthrax lethal factor protease by aminoglycosides Petr Kuzmic1 Lynne Cregar2 Sherri Z. Millis2 and Mark Goldman 1 BioKin Ltd Pullman WA USA 2 Hawaii Biotech Inc. Aiea HI USA Keywords aminoglycosides Bacillus anthracis inhibition lethal factor protease mechanism Correspondence P. Kuzmic BioKin Ltd 1652 South Grand Ave. Suite 337 Pullman WA 99163 USA Fax 1 509 3323493 Tel 1 509 3344131 E-mail pksci01@ Present address University of Hawaii at Manoa Cardiovascular Research Center Complementary and Alternative Medicine Honolulu HI 96822 USA Received 29 March 2006 revised 5 May 2006 accepted 10 May 2006 doi We report a detailed kinetic investigation of the aminoglycosides neomycin B and neamine as inhibitors of the lethal factor protease from Bacillus anthracis. Both inhibitors display a mixed-type noncompetitive kinetic pattern which suggests the existence of multiple enzyme-inhibitor binding sites or the involvement of multiple structural binding modes at the same site. Quantitative analysis of the ionic strength effects by using the Debye-Huckel model revealed that the average interionic distance at the point of enzyme-inhibitor attachment is likely to be extremely short which suggests specific rather than nonspecific binding. Only one ion pair seems to be involved in the binding process which suggests the presence of a single binding site. Combining the results of our substrate competition studies with the ionic strength effects on the apparent inhibition constant we propose that aminoglycoside inhibitors such as neomycin B bind to the lethal factor protease from B. anthracis in two different structural orientations. These results have important implications for the rational design of lethal factor protease inhibitors as possible therapeutic agents against anthrax. The strategies and methods we describe are general and can be employed to investigate in depth the mechanism

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